Interleukin 1 (IL-1p) is a 17.5 kDa polypeptide hormone synthesized and secreted by stimulated monocytes. The initial translation product of IL-1.beta. is a larger 31 kDa biologically inactive precursor polypeptide. The N-terminus of biologically active, mature IL-1.beta. derived from human activated monocytes has been characterized by an N-terminal amino acid sequence beginning with Ala-Pro. See, for example, European Patent Application EP-A 0165654 and March et al., Nature (London) 315:641-47 (1985) for sequence information of human IL-1.beta..
Many physiological actions and biological activities of IL-1 have been identified. IL-I biological activity is often determined by assaying for stimulation of thymocyte proliferation or by measuring interleukin-2 (IL-2) biological activity. IL-I activities include stimulation of B-lymphocyte maturation, lymphocyte proliferation, stimulation of fibroblast growth and induction of acute-phase protein synthesis by hepatocytes.
Other biological activities have been attributed to IL-1 polypeptides. These include control of differentiation and activation of lmphocytes, stimulation of lymphokine and prostaglandin production, promotion of inflammation, induction of acute phase proteins, stimulation of bone resorption, and alteration of the level of iron and zinc in blood. Moreover, it has recently been found that IL-1 can stimulate the hypothalamus-pituitary-adrenal axis, suggesting that IL-1 is integrated in the complex neuroendocrine network that controls homeostasis. This is further supported by the finding that administration of low doses of IL-I to normal mice results in both a several-fold elevation of glucocorticoid output and in a long-lasting blood glucose concentration increase.
The N-terminal Ala residue of human mature IL-1.beta. is in the 117 position and an Asp residue is in the 116 position counting from the N-terminus of human precursor IL-1.beta. polypeptide. Mature IL-1.beta. consists of the C-terminal 153 residues of the precursor polypeptide.
Maturation and release of mature IL-1.beta. from macrophages does not proceed by conventional means normally associated with most secretory proteins because the precursor IL-1.beta. polypeptide lacks a hydrophobic signal sequence. Further, IL-1.beta. is not associated with a membrane-bound compartment in monocytes. [Singer et al., J. Exp. Med. 167:389-407 (1988)]. Most secretory proteins are characterized by the presence of a hydrophobic stretch of amino acids called a signal sequence. The signal sequence directs the translocation of the protein across the membrane of the endoplasmic reticulum during protein synthesis. The protein is subsequently ushered out of the cell via exocytosis. Most secreted proteins have a signal sequence at the amino terminal that is removed upon translocation. Other proteins, such as ovalbumin, have an internal signal sequence that is not removed upon translocation. Both precursor forms of IL-1.beta. and IL-1.beta. (March et al.) lack any region (either amino terminal or internal) with sufficient hydrophobicity and length to qualify as a signal sequence.
A further indication of an unusual maturation pathway for IL-1.beta. is the absence of a pair of basic amino acids near the N-terminus of the mature polypeptide. The amino acid sequence Tyr-Val-His-Asp- precedes the N-terminal Ala-Pro of the mature human IL-1.beta. polypeptide. Moreover, Young et al., J. Cell Bio., 107:447-56 (1988) found that fibroblasts transfected with cDNA coding for precursor IL-1.beta. were unable to process the precursor polypeptide into mature IL-1.beta.. Instead, the transfected fibroblasts produced high levels of inactive precursor polypeptide. The results reported by Young et al. are consistent with other reports for other cell types, including T cells, epidermal cells and B cells.
Hazuda et al., J. Biol. Chem., 263:8473-79 (1988) have reported that both the precursor and mature forms of IL-1.beta. appear in the supernatants of activated monocytes with little or no preference. Hazuda et al. suggest that IL-1.beta. processing is "intimately coordinated" with secretion.
There have been several attempts to characterize or isolate the system responsible for processing IL-1.beta. from its translated precursor form to its active mature form. Black et al., J. Biol. Chem., 263:9437-42 (1988) [Black et al. I] suggest that the cleavage pattern of precursor IL-1.beta. is affected by myeloid cell membranes and results from the action of a plurality of proteases which act as an IL-1.beta. processing system. A subsequent article by Black et al., J. Biol. Chem., 264:5323-26 (1989) [Black et al. II] describes a single protease that cleaves IL-1.beta. between His.sup.115 and Asp.sup.116, one residue upstream form the N-terminal Ala.sup.117 of mature IL-1.beta.. Thus, the protease described in Black et al. II generates a form of IL-1.beta. one amino acid longer than the mature IL-1.beta. purified from monocyte cultures. Black et al. II suggests that there may be an aminopeptidase in human blood that removes the N-terminal asparate residue to complete the processing.
Kostura et al., Proc. Nat Acad Sci. USA, 86:5227-31 (1989) refers to a protease with a similar cleavage pattern but "qualitatively different" from the protease described in Black et al. II. The Kostura et al. protease is characterized as being located in cytosol of monocytic cells. However, Kostura et al. did not further define or isolate the responsible polypeptide.
Finally, Black et al., FEBS Lett., 247:386-90 (1989) [Black et al. III] refer to a protease that generates mature IL-1.beta. from the precursor polypeptide and is characterized by being inhibited by iodoacetate and N-ethylmaleimide. Black et al. III attempted to purify their protease approximately 500 fold by a process starting by freeze-thawing cell lysates from THP-1 cells (ATTC) four times. Black et al. III centrifuged the lysates for 20 minutes at 36,590.times.g. The supernatant was applied to a DEAE-Sephacel column equilibrated with 10 mM Tris-HCl (pH 8.1) and 5 mM dithiothreitol. The protease was eluted with 80-140 mM NaCl. The eluted material was diluted 1:5 with a buffer of 10 mM Tris-HCl (pH 8.1) and 5 mM dithiothreitol and applied to a procion red agarose column. The protease was eluted with 0.5-0.8 M NaCl, concentrated 20-fold in a Centriprep-10 concentrator, and then subjected to get filtration with Sephadex G-75. This procedure described in Black et al. III failed to demonstrate whether the protease activity was due to a single polypeptide or a group of processing enzymes.
Therefore, there is a need in the art to obtain the isolated system or single protease polypeptide responsible for processing precursor IL-1.beta. into its mature and biologically active form. The protease functions as an IL-1 agonist to increase IL-1 biological activity in vivo. Moreover, the isolated protease is useful for improving wound healing, treating arthritis, and treating or preventing the onset of autoimmune diseases, such as insulin dependent diabetes melitus, lupus disorders, Graves' disease, Hashimotos disease, and the detrimental side effects of radiation treatment.
Further, isolation and characterization of the protease responsible for processing precursor IL-1.beta. into its biologically active form aids in designing inhibitors for IL-1.beta. processing, because the availability of large quantities of IL-1.beta. pro serves as a useful screening vehicle for finding compounds having IL-1 antagonist activity. Such IL-1 antagonists or IL-1.beta. pro inhibitors are useful for treating inflammation and transplantation rejection.
A number of protease inhibitors specific for other protease activities have been described and reported in the literature. See, e.g, U.S. Pat. Nos. 4,644,055, 4,636,492 and 4,652,552. None of these previously reported protease inhibitors are specific for interleukin 1.beta. protease activity. None of the previously described protease inhibitors are effective in inhibiting the activity of interleukin 1 protease. Therefore, there is a need for a specific IL-1.beta. pro inhibitor that can prevent the cleavage of preIL-1.beta. into biologically active IL-1.beta.. Such an inhibitor can function as an IL-I antagonist. This invention provides IL-I antagonists that prevent the formation of biologically active IL-1.beta..